Human induced pluripotent stem cell engineering establishes a humanized mouse platform for pediatric low-grade glioma modeling

A major obstacle to identifying improved treatments for pediatric low-grade brain tumors (gliomas) is the inability to reproducibly generate human xenografts. To surmount this barrier, we leveraged human induced pluripotent stem cell (hiPSC) engineering to generate low-grade gliomas (LGGs) harboring the two most common pediatric pilocytic astrocytoma-associated molecular alterations, NF1 loss and KIAA1549:BRAF fusion. Herein, we identified that hiPSC-derived neuroglial progenitor populations (neural progenitors, glial restricted progenitors and oligodendrocyte progenitors), but not terminally differentiated astrocytes, give rise to tumors retaining LGG histologic features for at least 6 months in vivo. Additionally, we demonstrated that hiPSC-LGG xenograft formation requires the absence of CD4 T cell-mediated induction of astrocytic Cxcl10 expression. Genetic Cxcl10 ablation is both necessary and sufficient for human LGG xenograft development, which additionally enables the successful long-term growth of patient-derived pediatric LGGs in vivo. Lastly, MEK inhibitor (PD0325901) treatment increased hiPSC-LGG cell apoptosis and reduced proliferation both in vitro and in vivo. Collectively, this study establishes a tractable experimental humanized platform to elucidate the pathogenesis of and potential therapeutic opportunities for childhood brain tumors. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-022-01428-2.


Introduction
Low-grade gliomas (LGGs; World Health Organization grade 1 and 2 astrocytomas) are the most frequently occurring cancers of the central nervous system (CNS) in children [41], accounting for one third of all pediatric brain tumors [44]. The most common pediatric LGG is the grade 1 pilocytic astrocytoma (PA), which arises
RNA sequencing or methylation studies were not used to compare the humanized iNPC-LGGs with resected patient LGGs: iNPC-LGGs are composed entirely of human neoplastic cells and cannot be directly compared with human LGG biospecimens, in which 30-50% of the cells are monocytes, neurons, and T cells [13,23,50,51,57]. In addition, while methylation has proven valuable for separating classes of brain tumors, it is not as accurate in distinguishing subclasses of pediatric LGGs and glioneuronal tumors. This is due in part to the variable contributions of non-neoplastic elements, with the diagnostic standard involving histology, immunohistochemistry, and genetic analysis for the specific drivers involved (e.g., BRAF, NF1) [12,52]. For these reasons, we employed two additional complementary approaches to compare hiPSC-LGGs to their spontaneously arising clinical counterparts. First, leveraging human bulk RNA sequencing data [46], we identified PDPN as a gene differentially expressed both in NF1-associated and sporadic pilocytic astrocytomas relative to control non-neoplastic brain tissue (Additional file 1: Fig. S2e). Second, we found that FABP7, which we previously showed to be overexpressed in mouse optic gliomas relative to control optic nerve by bulk RNA sequencing [15], was increased both in NF1-associated and sporadic pediatric LGGs relative to control non-neoplastic human brain (Additional file 1: Fig. S2e). Similar to their spontaneously arising pediatric LGG counterparts, both PDPN and BLBP (FABP7 protein) were expressed in the humanized NF1-null and KIAA1549:BRAF-associated LGGs in situ (Fig. 1d).
As expected for pediatric low-grade tumors, and mirroring clinical observations, mice with hiPSC-derived LGGs did not exhibit increased mortality nor obvious abnormal neurologic findings. To determine whether these lesions reflected the chronic non-malignant nature of pediatric LGGs, we assessed tumors at 1, 3 and 6 mpi (Fig. 2). Using power calculations with a two-sided, two portions test, where power was set at 80% and significance at 0.05, we estimated that a minimum of 4, 4, and 5 mice would be required to detect tumor formation at 1 mpi, 3 mpi and 6 mpi, respectively. With appropriately powered cohorts, mice injected with NF1-null and KIAA1549:BRAF-expressing iNPCs formed LGGs that were histologically similar at 1, 3 and 6 mpi (Fig. 2a). While these lesions grew in size over time, occupying on average 10-36% of the brainstem or 10-15% of the cerebellum, respectively (Fig. 2b), they had similar proliferative indices (Ki67 + cells) at 1, 3 and 6 mpi (Fig. 2c). The glioma tumor areas exhibited low proliferative indices (4-6% Ki67 + cells), within the upper range of proliferation rates seen in pediatric PAs [64], whereas the embryonal-like areas exhibited slightly higher proliferative rates (8-15% Ki67 + cells) (Fig. 2a, c). There was no evidence of increased apoptosis (0.38-0.41% TUNEL + cells; Fig. 2d) or increased cellular senescence (0.3-0.4% β-galactosidase + cells; Additional file 1: Fig. S2f ) in vivo. Taken together, these findings demonstrate that iNPCs generate both NF1-associated and sporadic LGGs in vivo.

iNPC-LGG formation requires CD4 + T cell depletion
Similar to human patient brain tumor xenografts, iNPC lineage cells did not form LGGs following injection into wild type mice (Fig. 4a). To identify mouse strains that permit LGG formation, NF1-null iNPCs were injected into the brainstems of a series of immunodefective mouse strains (listed in Fig. 4a). Whereas LGGs readily formed in NOD/SCID, CD4-deficient and CD4/CD8-deficient mice at 1mpi, no tumors developed in CD8-deficient mice or strains deficient in the expression of microglia or T cell chemokine receptors (Cx3cr1, Ccr2) [45], components required for murine Nf1 optic glioma formation and growth (Additional file 1: Fig. S5a). To identify potential responsible molecular etiologies underlying CD4 + T cell deficiency-mediated LGG formation, we performed transcriptomal analysis on whole brainstems of wild type and Rag1 −/− mice (Fig. 4b, c; Additional file 1: Fig. S5b). We initially identified eight downregulated transcripts and one upregulated transcript in Rag1 −/− relative to wild type mouse brainstems (Fig. 4d). While three downregulated transcripts were validated in independentlyacquired Rag1 −/− brainstem samples (Chil3, Cd59, Cxcl10; Fig. 4e; Additional file 1: Fig. S5c), only Cxcl10 expression was decreased in all immunodefective mouse strains that permitted LGG formation.
Since Cxcl10, a cytokine belonging to the CXC chemokine family, is predominantly expressed by microglia and astrocytes [68], we next analyzed these cell types in uninjected wild type and immunodeficient mouse brainstems. Whereas microglia density and morphology was relatively unaltered in all uninjected mouse brains analyzed (Additional file 1: Fig. S6a), there were fewer GFAP + , EAAT2 + and ALDH1L1 + astrocytes in the brainstems of all immunodefective mouse strains that permitted LGG formation (Additional file 1: Fig.  S6b). This finding suggested that LGG formation may require a deficit in astrocytes. Consistent with an astrocyte defect, astrocytes isolated from Rag1 −/− mice had reduced Cxcl10 expression relative to wild type controls (86.3% reduction, Fig. 4f ). Importantly, incubation of Rag1 −/− astrocytes with activated wild type mouse T cell-conditioned medium (TCM) induced Cxcl10 protein production. The greatest increase in Cxcl10 protein production was observed after induction of Rag1 −/− astrocytes with TCM from CD4 + (24.4-fold), relative to CD8 + (6.5-fold), T cells (Fig. 4g). Taken together, these results indicate that reduced astrocytic Cxcl10 expression in glioma-bearing mouse strains likely reflects an absence of CD4 T cells, which induce Cxcl10 expression in astrocytes to hinder LGG growth in vivo.

Cxcl10 inhibits pediatric LGG formation
To determine whether Cxcl10 inhibits cell cycle progression or induces progenitor cell differentiation,  Fig. 5b). In this respect, 95-100% of the differentiated GFAP + cells were cleaved caspase-3-positive, and 83.3-88.8% of the total number of cells undergoing apoptosis were astrocytes (Fig. 5b).
Together, these results demonstrate that Cxcl10 induces astrocytic differentiation, as well as cell death, in vitro.
To demonstrate that stromal Cxcl10 is sufficient to inhibit LGG formation in vivo, NF1-null iNPCs and iGRPs engineered to ectopically express murine Cxcl10 were injected into the brainstems of Rag1 −/− mice. In contrast to vector-infected controls, no LGGs formed in mice following the injection of NF1-null iNPCs and iGRPs expressing murine Cxcl10 (Fig. 5c). Conversely, to establish that Cxcl10 is necessary to inhibit LGG formation, we transplanted NF1-null iNPCs and iGRPs into the brainstems of Cxcl10 −/− mice. At 1 mpi, 92% and 85% of the injected mice developed iNPC-and iGRPderived LGGs, respectively (Fig. 5d). These lesions were hypercellular, exophytic, and immunopositive for GFAP and OLIG2 expression, with 4.9% and 4.9% Ki67 + cells, respectively (Fig. 5e). Taken together, these data demonstrate that Cxcl10 is both necessary and sufficient to suppress LGG cell growth both in vitro and in vivo.

Human pediatric LGG cell lines develop LGGs in Cxcl10 −/− mice
To extend these findings to human PDX modeling, we leveraged two primary human PA cell lines from one patient with an NF1-PA (JHH-NF-PA; NF1 loss) and another with a sporadic PA (Res186). While these lines grow briefly in larval zebrafish (6 days), they do not form tumors in athymic nude mice over the course of 12 months [65]. Similar to the experiments using hiPSC neuroglial lineage cells, 5 × 10 5 PA cell lines were injected into brainstems of Rag1 −/− and Cxcl10 −/− mice. Whereas human pediatric PA cells did not form gliomas in wild type mice, both Rag1 −/− and Cxcl10 −/− mice developed LGGs at 1mpi and 6mpi. These LGGs were hypercellular with microcystic components, parenchymal with exophytic components, immunopositive for OLIG2, but mostly negative for GFAP, expression (Fig. 6). These lesions exhibited proliferative indices between 4.2 and 4.7% at 1mpi and 3.4-3.5% at 6 mpi. Together, these results establish Cxcl10 −/− mice as a tractable in vivo platform for human pediatric LGG modeling.

hiPSC-LGG growth is reduced following MEK inhibition
Finally, to provide a proof-of-principle demonstration that this humanized LGG platform could be used for preclinical drug evaluation, we assayed the ability of a MEK inhibitor (PD0325901) to block hiPSC-LGG growth beginning at 1 mpi in vivo. The treatment lasted a total of four weeks, a time frame we previously reported to inhibit Nf1 mouse low-grade optic glioma growth [34]. While Ku80 + iNPC-LGGs were still detectable in mice following PD0325901 administration, there was increased tumor cell apoptosis (TUNEL + cells), as well as reduced tumor cell proliferation (Ki67 + cells), relative to vehicle-treated LGGs (Fig. 7). In vitro PD0325901 treatment of NF1 null iNPCs, iGRPs and iOPCs also resulted in decreased cell proliferation and increased apoptosis (Additional file 1: Fig. S7), thus establishing this experimental platform for preclinical therapeutic studies.

Discussion
Building upon prior studies using hiPSC-derived 2D and 3D organoid cultures to study high-grade gliomas [1,35,39,53] and medulloblastoma [63], we developed a humanized xenograft platform to model sporadic and NF1-associated pediatric LGGs and elucidate the pathogenesis, cellular origins, and signaling pathway dependencies. Beyond the value of this system to PDX pediatric LGG future preclinical experimentation, this study raises several important points germane to human brain tumor pathobiology. First, leveraging hiPSC engineering for humanized tumor modeling represents an efficient system applicable to other low-grade nervous system tumor xenografts, which have been extremely challenging to establish in mice [28,30,49]. Relevant to pediatric LGGs, like NF1-PA and BRAF-driven PAs, the derivative tumor cells undergo oncogene-induced senescence [49] and display a senescence-associated secretory phenotype (SASP) in vitro unless provided with fibroblast conditioned medium and ROCK inhibition [65] or senolytic inhibitors [11]. The fragility of these PA tumor cell cultures likely reflects their profound stromal (tumor microenvironment) dependence, as well as their limited intrinsic self-renewal capacity [59], as demonstrated using genetically engineered mouse models and murine pediatric LGG explant systems. In these studies, Nf1 optic glioma growth in mice requires T cell and microglia support through the elaboration of critical cytokines and growth factors, as Nf1 optic glioma stem cells cannot form glioma-like lesions in mice lacking these stromal cells [22,47]. Similarly, KIAA1549:BRAF-expressing cerebellar stem cells cannot form tumors in mice lacking the T cell and microglia Ccr2 chemokine receptor [14]. Moreover, the SASP represents a cellular state characterized by the secretion of inflammatory cytokines and immune modulators, which may counter the pro-tumoral support provided by T cells and monocytes in the tumor microenvironment.
Second, the hiPSC-LGG explant system provides a tractable platform to define the putative cells of origin for histologically distinct tumors, as well as histologically similar tumors arising in different locations. This is particularly important with respect to the cellular ontogeny of brain cancers. In this regard, previous mouse modeling experiments have demonstrated that multiple cell types, including differentiated cells (astrocytes [6], neurons [17]), can give rise to high-grade gliomas. Using other murine modeling approaches, NPCs [37,45,60], or their derivative progenitor cells [2,8], have been shown to be the putative cells of origin, while restricted progenitors of the NPC lineage give rise to oligodendrogliomas [48] and proneural glioblastomas [40]. Moreover, additional cellular constraints, including the specific neuroglial progenitor population [60] and the particular brain location (third ventricle versus lateral ventricle germinal zone [36]) are critical determinants that dictate glioma formation and latency. In this report, we leveraged specific human neuroglial lineage cells to determine that not all lineages faithfully recapitulate LGG histological features. Whereas astrocytes do not give rise to these tumors, iGRPs, iOPCs and iNPCs with NF1 loss or KIAA1549:BRAF expression form LGGs within a month. It is intriguing to note that iGRPs give rise to more compact tumors with stronger GFAP than OLIG2 immunoreactivity, resembling optic pathway and brainstem gliomas, while the iOPC-derived lesions have looser stroma with microcystic changes and a higher density of OLIG2 + than GFAP + cells, similar to many cerebellar human PAs. In contrast, Olig2 + , NG2 neg glial progenitors form Nf1 optic gliomas with delayed latency [60], which may reflect innate differences between neuroglial cell populations in humans and rodents and/or different potential cells of origin [38,56].
Third, the finding that humanized pediatric LGGs develop in a mouse strain not lacking immune cells opens a new avenue for the in vivo study of low-grade PDXs. Whereas some orthotopically injected high-grade PDXs grow in wild type mice [25], the majority of PDX studies leverage immunocompromised (usually athymic and NOD/SCID) mice, due to their inherent inability to reject engrafted human cells. However, even in these immuneimpaired animals, not all tumors are able to grow [25,31], possibly due to the lack of a trophic environment provided by a complete immune system. Since T cells present in human PAs [13] and Nf1 murine low-grade gliomas [22] establish a supportive microenvironment for low-grade glioma growth in vivo, it is important to use host systems with immune systems with limited immunologic impairment, such as Cxcl10 −/− mice. Additional studies are in progress to define immune function in Cxcl10-deficient mice.
The relevance of Cxcl10 production to xenograft establishment and tumorigenesis is further strengthened by prior reports demonstrating that elevated CXCL10 in bronchoalveolar lavage fluid is associated with acute lung transplant rejection [26] and that viral CXCL10 gene therapy improves cervical cancer xenograft responses to radiotherapy [69]. In addition, increased Cxcl10 expression is inversely correlated with tumor growth and is associated with cardiac [24] allograft rejections in the PNS, such that enhanced systemic Cxcl10 expression is a prognostic biomarker of graft-versus-host disease [33]. Moreover, reduced Cxcl10 levels in HGG spheroids caused by long-term in vitro culture correlates with higher engraftment rate in immunocompetent rats [27]. Finally, CXCL10 is induced as part of the SASP [66], which limits primary human LGG cell growth in vitro through increased senescence [11,49]. Understanding the mechanisms underlying CXCL10-mediated hiPSCand patient-derived xenograft growth inhibition will be critical to the development of second-generation PDX models of pediatric LGGs.

Conclusions
It should be appreciated that this xenograft model system has some inherent limitations. Rag1 −/− mice have impaired immune systems and the resulting LGGs lack some of the non-neoplastic cell types (e.g., T cells) important for murine LGG growth [3,22,46,47]. Similarly, the impact of Cxcl10 loss on immune system function is unknown. Ongoing studies are focused on co-introducing hiPSC-derived monocyte populations, as well as defining the impact of Cxcl10 loss on non-neoplastic cell function. While additional work will be required to create preclinical models that fully recapitulate all of the elements of pediatric LGGs, we have generated an experimentally tractable in vivo platform for humanized orthotopic pediatric LGG modeling. Further refinement of this model has great potential to galvanize the study of a variety of different types of brain and nerve tumors, including previously unexplored low-grade subtypes, as well as advance our understanding of the molecular and cellular origins of these common brain tumors in children.

Study approval
All experiments were performed under active and approved Animal Studies Committee protocols at Washington University.

Animals
Mice were maintained on a 12 light/ dark cycle in a barrier facility, had ad libitum access to food and water, were exclusively used for the purposes of this study, and had not undergone any additional procedures. Mating cages housed one male and two females of the same genotype, and males were separated from dams at the time of delivery. Injections were performed on entire litters of 0-3-day-old mice of the following strains: C57BL/6J, Rag1 −/− (B6.129S7-Rag1 tm1Mom /J; strain 002216, Jackson laboratories), Cxcl10 −/− (B6.129S4-Cxcl10 tm1Adl /J; strain 006087, Jackson laboratories), CD4-deficient  [47] mice. Injected pups were allowed to recover and were subsequently immediately returned to their maternal cage until weaning. Mice of both sexes were randomly assigned to all experimental groups without bias, and the investigators were blinded until final data analysis during all of the experiments.

Intracranial injections
Postnatal day 0-4 animals were anaesthetized and intracranially injected in accordance with active Animal Studies Committee protocols at Washington University. 1 × 10 4 , 5 × 10 4 , 1 × 10 5 or 5 × 10 5 cells resuspended in 2 µL ice-cold PBS were injected 0.7 mm to the right of the midline into either the midbrain (0.5 mm posterior to Lambda; 2 mm deep), or the cerebellum (2 mm posterior to Lambda; 1 mm deep) of neonatal mice using a Hamilton syringe. Animals were aged to 1, 3 or 6 months postinjection prior to tissue harvesting and analysis.

PD0325901 treatments
Twenty 4-week-old Rag1 −/− mice of both sexes harboring NF1-null iNPC-LGGs 1 month post-injection were intraperitoneally injected either with 10% DMSO in saline or with 5 mg/kg/day PD0325901 (Sigma-Aldrich), 6 days a week, for a total of four weeks [34]. Treated mice were collected for histopathologic analysis. For in vitro experiments, iNPCs, iGRPs and iOPCs were treated with 10 nM PD0325901 for 24 h prior to immunocytochemical analysis.

Magnetic resonance imaging (MRI)
Injected mice were transcardially perfused with Ringer's solution and 4% paraformaldehyde (PFA). The entire brain was removed from the animals and post-fixed in 4% PFA overnight prior to rehydration in phosphate buffered saline (PBS) for a minimum of 7 days. Rehydrated brain samples underwent MRI. Each mouse brain was then packed into a 2 mL plastic vial, supported with fiberglass and immersed in

Tissue fixation and immunohistochemistry
Injected mice were transcardially perfused at 1, 3 or 6 months post-injection, initially with Ringer's solution, and then followed by ice-cold 4% paraformaldehyde. Whole mouse brains were harvested, post-fixed in 4% PFA and dehydrated in 70% ethanol. Dehydrated samples were then paraffin-embedded and serially sectioned (5 µm). Hematoxylin and eosin (H&E), as well as antibody immunohistochemical staining, were performed using the primary antibodies, Vectastain ABC kit (Vector Laboratories) and appropriate biotinylated secondary antibodies provided in Additional file 1: Table S1. Immunostaining was performed using the primary antibodies listed in Additional file 1: Table S1 with appropriate Alexa Fluor-conjugated secondary antibodies. Bis-benzamide (Hoechst) was used as a nuclear counterstain.

Immunocytochemistry
Immunocytochemistry was performed on hiPSCs, iNPCs, iGRPs, iOPCs and astrocytes using the primary antibodies described in Additional file 1: Table S1. Briefly, adherent cells were washed, fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, before blocking in 10% goat serum and incubation overnight at 4 °C in primary antibodies diluted in 2% goat serum at the manufacturer's suggested concentrations. Appropriate secondary Alexa-Fluor-conjugated secondary antibodies diluted to 1:200 were employed and bis-benzamide (Hoechst) was used as a nuclear counterstain. Cells were mounted with Immu-mount (Fisher) and imaged on a Leica DMi1 fluorescent microscope using the Leica LAS X software, as per manufacturer's instructions.

BrdU proliferation, RAS activity, cAMP and Cxcl10 ELISA
RAS activity (ThermoFisher), cAMP (Fisher) detection, BrdU proliferation assays (Roche) and direct cell counting were performed according to the manufacturer's instructions. Cxcl10 (Ray Biotech) ELISA assays were performed according to the manufacturer's instructions. Each assay was performed using a minimum of three independently generated biological replicates.

Lentivirus production, cell infection and CXCL10 peptide treatment
Cxcl10 cDNA (Sino Biological) and lentiviral packaging vectors, or KIAA1549:BRAF adenoviral DNA, were transfected in HEK293T cells using Fugene HD (Promega) following the manufacturer's instructions. Viral supernatants were collected 48 h post-transfection, filtered and used to directly infect iNPCs and iGRPs for 24 h. Cxcl10-GFP expression was confirmed by Western blotting as previously described (Anastasaki et al., 2015). KIAA1549:BRAF-infected cells were puromycin-selected for 2 weeks prior to further expansion. Infected cells were used for intracranial injections in Rag1 −/− neonatal mice.
For cell proliferation, cell death and differentiation analyses, iNPCs were treated with 25 or 100 pg/mL of murine recombinant Cxcl10 (PeproTech) for 24 h. Each experiment was performed a minimum of three times on independently generated samples.  Table S2) and a commercially available Taqman mastermix (Thermo Fisher Scientific) following the manufacturer's instructions. Relative transcript expression was calculated using the ΔΔCT analysis method and normalized to Gapdh as an internal control following the manufacturer's instructions (ThermoFisher). For RNA sequencing, total RNA from three C57BL/6J and three Rag1 −/− brainstem samples was submitted to Washington University Genome Technology Access Center (GTAC). Samples were prepared according to the library kit manufacturer's protocol, indexed, pooled, and sequenced on an Illumina HiSeq. Basecalls and demultiplexing were performed with Illumina bcl2fastq software and a custom python demultiplexing program with a maximum of one mismatch in the indexing read. The analysis was generated using Partek Flow software, version 8.0. RNA sequencing reads were aligned to the mm10-RefSeq Transcripts 83 assembly with STAR version 2.5.3a. Gene counts and isoform expression were derived from the annotation model output. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. Normalization size factors were calculated for all gene counts by CPM to adjust for differences in library size. Gene specific analysis was then performed using the lognormal with shrinkage model (limma-trend method) to analyze for differences between Rag1 −/− and control C57 mice. The results were filtered for only those genes with p values ≤ 0.05 and log foldchanges ≥ ± 2. Principal component analysis was conducted in Partek Flow using normalized gene counts. RNA sequencing data have been deposited in the GEO portal (GSE174624).

T cell isolation and culture
4-6-week-old C57BL/6J and Rag1 −/− mouse spleens were homogenized into single cell suspensions by digestion in PBS containing 0.1% BSA and 0.6% sodium citrate. The homogenates were subsequently washed and incubated with 120 Kunitz units of DNase I for 15 min following red blood cell lysis (eBioscience). Cells were then filtered through a 30 µM cell strainer to obtain a single cell suspension. CD4 + and CD8 + T cells were isolated using CD8a (Miltenyi Biotech) or CD4 (Miltenyi Biotech) T cell isolation kits, respectively. T cells were maintained at 2.5 × 10 6 cells ml −1 in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/ streptomycin. T cells were activated by 1.25 µg ml −1 anti-mouse CD3 (Fisher Scientific) and 2 µg ml −1 anti-mouse CD28 (Fisher Scientific) antibody treatment for 48 h. T cell conditioned media (TCM) was collected both from nonactivated and activated T cells following 22 µM filtration for subsequent chemokine assay, ELISA and co-culture experiments.

Astrocyte isolation and culture
4-to 6-week-old C57BL/6 J and Rag1 −/− mice were transcardially perfused with DPBS and whole brains collected following the removal of the cerebellum and olfactory bulbs. A single cell suspension was generated using the Miltenyi Biotech adult brain dissociation kit following the manufacturer's instructions. The resulting cells were seeded in Poly-l-Lysine-coated (Millipore Sigma) T75 flasks and incubated in Minimal Essential Medium supplemented with 1 mM l-glutamine, 1 mM sodium pyruvate, 0.6% D-(+)-glucose, 100 μg ml −1 penicillin/ streptomycin and 10% FBS. A complete media change was performed after 24 h and every third day after that for 12-14 days, in order to obtain mixed glial cultures composed of microglia on an astrocyte monolayer. To release the microglia, flasks were mechanically shaken at 200 rpm for 5 h at 37 °C, and the microglia-containing supernatant discarded. Astrocyte-enriched cultures containing > 95% GFAP-positive astrocyte monolayers were passaged with 0.1% trypsin (Invitrogen) for subsequent experiments. Conditioned media from naïve or TCM-treated astrocytes was collected following 22 µM filtration.

Quantification and statistical analysis
All statistical tests were performed using GraphPad Prism 5 software. 2-tailed Student's t-tests, or one-way analysis of variance (ANOVA) with Bonferroni post-test correction using GraphPad Prism 5 software. Statistical significance was set at p < 0.05, and individual p values are indicated within each graphical figure. A minimum of 3 independently generated biological replicates was employed for each of the analyses. Numbers (n) are noted for each individual analysis.